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Immunochemical detection of human plasma enzyme lecithin:cholesterol acyltransferase Hon, Kenneth K. 1981

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IMMUNOCHEMICAL DETECTION OF HUMAN PLASMA ENZYME LECITHIN:CHOLESTEROL ACYLTRANSFERASE by KENNETH K. HON B. S c , The University of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1981 (S) Kenneth K. Hon., 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o,f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D E - 6 ( 2 / 7 9 ) Abstract A simple and s p e c i f i c electroimmunoassay has been developed for the human plasma enzyme l e c i t h i n : c h o l e s t e r o l acyltransferase (LCAT; E.C. 2.3.1.43). This enzyme catalyzes the transfer of an acyl group from the C:2 p o s i t i o n of l e c i t h i n to the 3-hydroxyl group of free cholesterol to form cholesterol ester and l y s o l e c i t h i n . The i n i t i a l rate of this reaction i s commonly measured by various radiochemical techniques and serves to r e f l e c t the enzyme l e v e l i n plasma. However, such measurement of the enzymatic a c t i v i t y f a i l s to separate the influences of enzyme, substrate and cofactor on the e s t e r i f i c a t i o n rate. A d i r e c t measurement of the protein mass through an immunoassay coupled with a determination of enzymatic a c t i v i t y may help to provide information that would allow d i f f e r e n t i a t i o n of enzyme abnormalities from that of other fa c t o r s . The electroimmunoassay described i n this thesis involved the production of a mono-s p e c i f i c antibody against the enzyme and the subsequent use of an immuno-electrophoretic procedure. Electrophoresis was performed i n an agarose g e l containing anti-LCAT antibody. Quantitation of LCAT protein l e v e l was based on measurement of the area of the p r e c i p i t a t e (rocket) i n the samples and the pure LCAT standards. S p e c i f i c i t y of the antibody produced was assessed as follows: ( i ) the antibody formed a single p r e c i p i t i n arc with both p u r i f i e d LCAT enzyme and normal human plasma, ( i i ) the antibody showed no reaction with most of the major lipoproteins very low density l i p o p r o t e i n s , low density lipoproteins and high density l i p o p r o t e i n s . - i i i -( i i i ) complete i n h i b i t i o n of LCAT a c t i v i t y was achieved when the antibody was incubated with e i t h e r human plasma or p u r i f i e d enzyme, (iv) the antibody did not react with plasma of patients with LCAT def i c i e n c y . (v) pre-incubation of antibody with normal human plasma led to a s i g n i f i c a n t decrease i n the antibody's ant^-LCAT a c t i v i t y i n a subsequent assay. No such decrease, however, was observed a f t e r s i m i l a r immunoabsorption of the antibody with LCAT d e f i c i e n t plasma. The concentration (mean + S.D.) of LCAT protein i n normal subjects was 5.40 + 0.54 mg/1 (n = 12). Two patients with in h e r i t e d LCAT deficiency also showed no detectable l e v e l of the enzyme i n t h e i r plasma. An in v e s t i g a t i o n of the family members revealed that the patients' parents and two of t h e i r s i b l i n g s had sub-normal amount of LCAT enzyme (3.46 + 0.48 mg/1, n = 4). Their enzymatic a c t i v i t i e s , as measured by an a r t i f i c i a l substrate assay (phosphotidylcholine:free c h o l e s t e r o l liposomes), were also lower than normal (26-37 versus 45-85 nmole free cholesterol e s t e r i f i e d / m l plasma/hr.). This immunoassay appears to be a suitable procedure for the quantitation of plasma LCAT. Moreover, this quantitation correlated well with the enzyme a c t i v i t y i n a study of the LCAT d e f i c i e n t family (r = 0.93, n = 6). - i v -TABLE OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Tables v L i s t of Figures v i L i s t of Abbreviations v i i Acknowledgement v i i i Introduction ( i ) H i s t o r i c a l background 1 ( i i ) Plasma lipoproteins 1 ( i i i ) Lipoprotein substrates for LCAT reaction 6 (iv ) Role of the reaction 9 (v) F a m i l i a l LCAT deficiency 13 (vi) Methods of LCAT assay 16 Materials and Methods ( i ) Antigen preparation 20 ( i i ) Immunization 22 ( i i i ) Characterization of antibody 23 (iv) Immunoassay 26 (v) Protein determination 27 Results f Characterization of rabbit anti-LCAT serum: 29 (A) Immunodiffusion (B) Immunoinhibition of LCAT a c t i v i t y Immunoassay of human plasma LCAT 47 Discussion , 58 References 63 - v -LIST OF TABLES Table Page I Human plasma lipoproteins 4 II D i s t r i b u t i o n of apoproteins i n human plasma lipoproteins 5 III Major l i p i d s of plasma lipoproteins i n patients with f a m i l i a l 15 LCAT deficiency IV P u r i f i c a t i o n of l e c i t h i n - c h o l e s t e r o l acyltransferase 30 V - l I n h i b i t i o n of plasma LCAT a c t i v i t y 38 V-2 E f f e c t of immunoglobulin on LCAT a c t i v i t y 39 VI Time course p r o f i l e 41 VII-1 E f f e c t of LCAT d e f i c i e n t plasma on anti-LCAT serum 45 VIII-1 I n h i b i t i o n of p u r i f i e d LCAT enzyme 52 VIII-2 I n h i b i t i o n of p u r i f i e d LCAT enzyme as a function of time 53 IX Quantitation of plasma LCAT by 'rocket' Immunoelectrophoresis 54 X Plasma l i p i d s and LCAT values i n family with LCAT deficiency 57 - v i -LIST OF FIGURES Figure Page 1 The major reactants involved i n the reaction catalyzed by human 2 plasma l c e i t h i n : c h o l e s t e r o l acyltransferase. 2 Mechanism for the formation of H D L 3 and HDL2 p a r t i c l e s as 8 proposed by Eisenberg. 3 Role of LCAT i n the homeostasis of cho l e s t e r o l i n lipoproteins 10 and c e l l membranes. 4. The catabolic pathways of lipoproteins i n human. 12 5 DEAE-Sephacel column chromatography of the dialysed 31 d 1.21-1.25 g/ml f r a c t i o n . 6 Hydroxylapatite column chromatography of the active DEAE- 32 sephacel eluates. 7 Polyacrylamide gel electrophoresis of the p u r i f i e d enzyme. 33 8a Immunodiffusion of anti-LCAT antiserum against human plasma 34 and p u r i f i e d LCAT preparation. 8b Radial immunodiffusion 35 9 Immunoinhibition of plasma LCAT a c t i v i t y 40 10a Time course p r o f i l e on i n h i b i t i o n of plasma LCAT a c t i v i t y by 42 anti-LCAT antiserum. 10b Percent i n h i b i t i o n of plasma LCAT a c t i v i t y by anti-LCAT 43 antibody as a function of time. 11 E f f e c t of LCAT-deficient plasma on anti-LCAT a c t i v i t y of 46 antiserum. 12 Time course p r o f i l e . 49 13 Immunoinhibition of p u r i f i e d LCAT a c t i v i t y i n an a r t i f i c i a l 50 substrate system by anti-LCAT antibody. 14 Standard curve for the determination of plasma protein l e v e l 51 of LCAT. 15 "Rocket" Immunoelectrophoresis of l e c i t h i n : c h o l e s t e r o l 55 acyltransferase. 16 Quantitation of plasma LCAT i n family members of LCAT d e f i c i e n t 56 patients by electroimmunoassay. - v i i -LIST OF ABBREVIATIONS LCAT - L e c i t h i n : c h o l e s t e r o l acyltransferase TG - T r i g l y c e r i d e s VLDL - Very Low Density Lipoprotein LDL - Low Density Lipoprotein HDL - High Density Lipoprotein PC - Phosphotidylcholine FC - Free Cholesterol CE - Cholesterol Ester LPL - Lipoprotein Lipase Apo - Apoprotein - v i i i -Acknowledgement I wish to thank my supervisor, Dr. J . F r o h l i c h , for his advice and encouragement throughout the course of my work. His understanding and patience w i l l always be deeply appreciated and remembered. The i n t e r e s t and help he has shown have made this project possible. I am also g r a t e f u l l y indebted to Mr. Roger McLeod for his expert experimental assistance i n the laboratory. His r e l e n t l e s s e f f o r t i n providing me with the much needed enzyme i s indispensable. I would also l i k e to thank Mr. Chuck Yeung for his assitance i n bleeding the r a b b i t s . His f r i e n d l y and unique comments also help to make the laboratory atmosphere a joy to work i n . The t i r e l e s s e f f o r t of Miss Sandra Sturgeon i n typing and preparing this thesis i s also g r a t e f u l l y appreciated. - 1 -INTRODUCTION H i s t o r i c a l background The e s t e r i f i c a t i o n of ch o l e s t e r o l i n plasma was f i r s t noted by Sperry i n 1935 (1). He showed that maximal e s t e r i f i c a t i o n of cholesterol occurred when human plasma was incubated at 37°C, pH 8. The reaction could be stopped by heating the plasma at 60°C. Sperry postulated that the cholesterol e s t e r i f i -cation was an enzymatic reaction involving a plasma l e c i t h i n a s e ( l e c i t h i n as a source of fat t y acid) and a c h o l e s t e r y l ester hydrolase. However, i n 1962 Glomset presented evidence that only one enzyme was responsible for the e s t e r i f i c a t i o n of ch o l e s t e r o l i n plasma (.2). This enzyme transferred an acyl group from the C:2 p o s i t i o n of l e c i t h i n (PC) to free cholesterol (FC) and l y s o l e c i t h i n and c h o l e s t e r y l esters {CE) were formed. The enzyme was named l e c i t h i n : c h olesterol acyltransferase (.LCAT; E. C .2 .3 .1.43 ; . The reaction catalysed by LCAT i s shown in F i g . 1. Although the LCAT reaction involves the f a t t y acid i n C:2 p o s i t i o n of phospholipid, t h i s is not an absolute s p e c i f i c i t y {2,11). Up to 40% of f a t t y acids i n the C : l po s i t i o n have been shown to be released depending upon the degree of unsaturation and chain length of fatty acids at the two po s i t i o n s . The molecular weight of the enzyme as determined by sedimentation equilibrium i s 59,000 (56), whereas higher values of 65-70,000 are reported from SDS polyacrylamide gel electrophoresis studies (66). Plasma lipoproteins Both c h o l e s t e r o l (free and e s t e r i f i e d ) and l e c i t h i n occur i n plasma as major constituents of li p o p r o t e i n s . Thus the LCAT reaction plays a major role - 2 -LECITHIN C H 2 0 —saturated fatty acid C H O - u n s a t u r a t e d fatty acid 0 II C H 2 0 - P - c h o l i n e I OH CHOLESTEROL LECITHIN: CHOLESTEROL A C Y L T R A N S F E R A S E LYSOLECITHIN C H 2 0 - s a t u r a t e d fatty acid CHOLESTERYL ESTER CHOH 0 C H 2 0 - P - c h o l i n e OH unsaturated fatty acid F i g . 1. The major reactants involved i n the reaction catalyzed by human plasma l e c i t h i n r c h o l e s t e r o l acyltransferase (.Glomset, 1971). - 3 -in l i p o p r o t e i n metabolism. Lipoproteins are l i p i d - p r o t e i n complexes functioning primarily as c a r r i e r s of l i p i d (.especially of t r i g l y c e r i d e s (TG) and cholesterol.). Lipoproteins can be separated according to t h e i r s i z e s , charges and d e n s i t i e s . Table I shows composition of four basic classes of l i p o p r o t e i n s : chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (.LDL) and high density lipoproteins (.HDL). A major difference among these lipoproteins i s t h e i r l i p i d to protein r a t i o which determines the i r density. These differences i n densities have allowed the separations of lipoproteins by u l t r a c e n t r i f u g a t i o n . A l l lipoproteins possess s i m i l a r fundamental structure with a nonpolar core (.TG or CE) surrounded by a polar surface layer (.PC, FC and proteins) acting as a s t a b i l i z e r . As shown in Table I, the l i p i d composition of various lipoproteins is quite d i f f e r e n t . TG constitute most of the core of chylo-microns and VLDL whereas CE are the major core material in LDL and HDL. In human plasma most of the cholesterol e x i s t s i n e s t e r i f i e d form. About 2/3 of plasma CE are found i n LDL, 1/4 i n HDL and the rest i n VLDL. This difference i n the core component suggests that VLDL and chylomicrons are more important i n TG transport while LDL and HDL are mainly involved in cholesterol transport. The surface protein moiety of various lipoproteins is also d i f f e r e n t (Table II). Apo-proteins B and C predominate in VLDL and LDL, whereas apoprotein A is the major protein component in HDL. Recent development of better methods for i s o l a t i o n of these proteins from l i p i d has permitted better understanding of the s i g n i f i c a n c e of apoproteins in l i p o p r o t e i n metabolism. It i s now accepted that apo A-I i s an activator of LCAT (3,4) while apo-C II i s e s s e n t i a l for l i p o p r o t e i n lipase (LPL) a c t i v i t y (5). Although the functions of apo B and E are not f u l l y established, accumulating evidence suggests that TABLE I. HUMAN PLASMA LIPOPROTEINS Class Diameter (A) Density (g/ml) Electrophoretic Composition mobility (% dry mass) Core Surface TG CE Cholesterol Phospho- Protein l i p i d s Chylomicrons 800-5000 0.93 o r i g i n 86 3 2 7 2 VLDL 300-800 0.95-1.006 pre-6 55 12 7 18 8 LDL 216 1.019-1.063 6 6 42 8 22 22 HDL 2 100 1.063-1.125 a 5 17 5 33 40 HDL3 75 1.125-1.210 a 3 13 4 25 55 - 5 -TABLE I I . DISTRIBUTION OF APOPROTEINS IN HUMAN PLASMA LIPOPROTEINS3 Class Molecular weight Approx. concn Cmg/dU Composition in MAJOR LIPOPROTEIN (% Dry Mass) CLASSES Chylomicrons VLDL LDL HDL A-1 28,300 130 7 1 Trace 64 A-II 17,400 40 5 - Trace 20 B 10,000-250,000* 80 19 36 95 -C-I 7,000 6 11 3 Trace 6 C-II 10,000 3 15 7 Trace 1 C-II I 9,300 12 41 40 Trace 4 E 33,000 5 13 5 2 D 22,100 10 - Trace Trace 3 *M.W. for apo-B i s con t r o v e r s i a l aData compiled from references 14 and 76 - 6 -they may play a major role i n the secretion and biosynthesis of lipoproteins and are also e s s e n t i a l for receptor recognition and thus for the hepatic and peripheral c e l l u l a r uptake of lipoproteins (6-8). In summary, each of the four major classes of lipoproteins plays an unique role in the transport processes of l i p i d s i n plasma. The s p e c i f i c apoproteins of each l i p o p r o t e i n not only govern the i n t e r a c t i o n between lipoproteins themselves but also mediate reaction with enzymes and s p e c i f i c receptors on c e l l u l a r surfaces. Lipoprotein substrates for LCAT reaction Even though TG r i c h p a r t i c l e s such as VLDL and chylomicrons can increase the rate of LCAT reaction (9) HDL has long been recognized as the best sub-strate for the enzyme (10,11). This observation is not s u r p r i s i n g since the major protein moiety i n HDL i s apo-A I required for maximal LCAT a c t i v i t y . HDL is believed to be synthetized i n the l i v e r and secreted into the plasma as a nascent HDL molecule, a b i l a y e r disk p a r t i c l e composed mainly of apoprotein E, l e c i t h i n and free c h o l e s t e r o l . These nascent HDL p a r t i c l e s , i n i t i a l l y discovered i n rat l i v e r perfusate (12), resembled the abnormal HDL found i n patients with f a m i l i a l LCAT deficiency (to be discussed i n d e t a i l l a t e r ) . Whether these nascent p a r t i c l e s are primarily secretory products of l i v e r c e l l s or are also formed during catabolism of VLDL i s not known. However, i t was suggested that nascent HDL i s a better substrate for LCAT (12) than the "mature" HDL which represents an end product of the reaction (14J. This can be explained by the fact that nascent HDL p a r t i c l e s contain r e l a t i v e l y more l e c i t h i n and free cholesterol but less c h o l e s t e r y l ester which i s known to i n h i b i t the enzyme reaction (15). I t must be stressed that the above discussion is based mainly on r e s u l t s from animal (rat) experiments. Data on - 7 -normal human nascent HDL molecules are rare. Recently, Eisenberg presented (.in vitro,) experimental evidence which suggested that HDL i s the f i n a l metabolic product of VLDL l i p o l y s i s (.13,). It has been noted before that when properly i s o l a t e d by zonal centrifugation HDL of human plasma can be separated into two subfractions: HDL^ (density = 1.10 gm/mi; and HDL3 (d = 1.15 g/ml,) (16). Both of these molecules are spherical i n shape and have no major differences i n apoprotein content. In contrast the amount of both phospho-l i p i d s and t o t a l c h o l e s t e r o l is much higher i n HDL 2 than i n HDL^. More importantly, the HDL 2 f r a c t i o n contains a higher concentration of CE, making i t a more e f f i c i e n t c a r r i e r of cho l e s t e r o l . Using the above data Eisenberg proposed a hypothesis for the formation of the HDL molecule and i t s interactions with LCAT (13j. As a nascent HDL p a r t i c l e (or HDL precursor) i s f i r s t released into c i r c u l a t i o n , i t i s acted upon by LCAT and converted into the sph e r i c a l HDL^ molecule. This reaction is f a c i l i t a t e d by apo A-I proteins which are probably released from large lipoproteins a f t e r l i p o l y s i s by LPL. The HDL^ molecule c i r c u l a t e s i n the plasma u n t i l a s i t u a t i o n arises when more phospholipids and cholesterol are present i n the plasma (e.g. increase i n dietary uptake). In such a s i t u a t i o n the HDL^ molecule w i l l be transformed into HDL 2 through further reaction with apo AI and LCAT (Fig. 2). This proposed mechanism of formation of HDL molecules is based on a few i n i t i a l experiments and more rigorous testing of this hypothesis is needed before i t can be confirmed. However, Eisenberg's proposal is compatible with many c l i n i c a l and experimental observations. For example, HDL 2 is noted to be absent from the plasma of patients with l i p o p r o t e i n l i p a s e deficiency or apo C-II deficiency (17,18), suggesting that VLDL i s involved i n HDL formation. - 8 -CHYLi VLDL IDL. LDL LPL HDL-3 HDL-2" CHYL/ VLDL fDL, LDL L P L HDL-2 Apo A HDL-3 HDL-3 L C A T F i g . 2 . Mechanism for the formation of HDL3 and HDL2 p a r t i c l e s as proposed by Eisenberg (13,). The formation of the p a r t i c l e s are believed to be dependent on LCAT a c t i v i t y (" " = precursors). - 9 -Role of the reaction Since Glomset described the mechanism of LCAT reaction i n 1962 i t has been well established that i t is responsible for the formation of most of the c h o l e s t e r y l esters i n human plasma. Yet the exact p h y s i o l o g i c a l r o l e that the enzyme plays i n l i p o p r o t e i n metabolism i s s t i l l not f u l l y understood. However an impressive increase i n our knowledge of ch o l e s t e r o l metabolism makes i t possible to construct a p l a u s i b l e model of LCAT involvement i n li p o p r o t e i n metabolism. In the early 1970's, Glomset postulated that the major role of the LCAT reaction was to generate plasma c h o l e s t e r y l esters. He noted that c h o l e s t e r y l esters are a transport form of ch o l e s t e r o l and thus LCAT could a f f e c t trans-port of free cholesterol from peripheral tissues to the l i v e r CIO). This postulate was based on evidence that exchange of ch o l e s t e r o l existed between lipoproteins and plasma membranes and l a t e r received further support from the study of patients with f a m i l i a l LCAT deficiency (20-22). In 1970 another possible major function of LCAT was postulated by Schumacher and Adams (23). They suggested that LCAT was e s s e n t i a l i n the removal of "excess" surface l i p i d s (FC and PC) that were present a f t e r removal of TG from the chylomicrons and VLDL by the l i p o p r o t e i n lipase system. Glomset and Norum (21) combined the two hypotheses together in 1973 and suggested a broader view of the LCAT reaction which can be best i l l u s t r a t e d by th e i r own schematic diagram ( F i g . 3). The diagram shows the role of LCAT i n the homeostasis of ch o l e s t e r o l in lipoproteins and c e l l membranes. The HDL-LCAT system plays a c e n t r a l role i n the regulation of free cholesterol i n chylomicrons, VLDL, LDL and c e l l u l a r plasma membranes. The two authors believed that exchange of FC is non-enzymatic and may r e s u l t i n a net transfer - 10 -F i g . 3. Role of LCAT i n the homeostasis of cho l e s t e r o l i n lipoproteins and c e l l membranes (Glomset and Norum, 1974). - 11 -of free c h o l e s t e r o l depending on LCAT a c t i v i t y and a v a i l a b i l i t y of free c h o l e s t e r o l i t s e l f . With the recent better understanding of TG transport and of the formation of LDL from VLDL, a more detailed picture of LCAT involvement i n plasma l i p i d transport can be constructed. Figure 4 (Havel et a l . (14)) represents the current views on the i n t e r a c t i o n of human lip o p r o t e i n s . The two major enzymes involved i n the catabolic pathways are l i p o p r o t e i n lipase and LCAT. As VLDL and chylomicrons enter the c i r c u l a t i o n t h e i r inner core content of TG is continually hydrolyzed by the action of the lipase y i e l d i n g remnant (TG-poor) l i p o p r o t e i n p a r t i c l e s . As the TG core of these p a r t i c l e s i s further depleted, t h e i r surface c h o l e s t e r o l and phospholipids i n t e r a c t with LCAT i n the presence of HDL to form CE. The CE thus formed ultimately moves into the inside of the molecule to replenish the diminishing content of "core" l i p i d s . These reactions provide the needed surface-core r a t i o to s t a b i l i z e the molecule, and allow for conversion of the TG r i c h p a r t i c l e s into c h o l e s t e r o l - r i c h LDL (27-30). This pathway also represents one possible way of transfer of CE from HDL to LDL. Previously CE was shown to transfer d i r e c t l y from is o l a t e d HDL to LDL and VLDL upon incubation i n v i t r o (26). The transfer is mediated by a ch o l e s t e r o l ester transfer factor (19,43). This view of the LCAT involvement i n l i p o p r o t e i n metabolism i s consistent with observations i n LCAT d e f i c i e n t patients (31-33). Abnormally large VLDL and LDL p a r t i c l e s can be found i n the c i r c u l a t i o n of these patients with l i t t l e or no CE i n them. Yet when the patients' plasma is incubated with p u r i f i e d LCAT, the concentration of these abnormal size p a r t i c l e s is reduced with a concurrent increase i n normal LDL p a r t i c l e s (32,33). Of interest is that most of the CE thus formed are found to be associated with the VLDL and Bile Acids LOL Receptor Scavenger Pathway Pathway Phospholipids F i g . 4. The catabolic pathways of li p o p r o t e i n s i n human. I t i s through these pathways that both t r i g l y c e r i d e s and chol e s t e r o l are transported i n man. The two major enzymes that are involved are l i p o p r o t e i n l i p a s e (LPL) and l e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT) (Havel et a l . , 1980 (14))/ - 13 -LDL f r a c t i o n s . Results from i n v i t r o experiments support the mechanism outlined i n Figure 4 since s u b s t i t u t i o n of d e f i c i e n t serum with p u r i f i e d LCAT may well p a r a l l e l the s i t u a t i o n i n normal plasma where LPL and LCAT regulate plasma l i p o p r o t e i n metabolism. F a m i l i a l LCAT deficiency Many of the hypotheses regarding LCAT action presented above have been confirmed by studies of patients with f a m i l i a l LCAT deficie n c y . Before the discovery of these patients i n Norway i n 1966 (.34,35), i n v i t r o study of the LCAT reaction always presented problems since cholesterol i n human l i p o -proteins i s present mostly i n e s t e r i f i e d form. Studies of LCAT d e f i c i e n t patients, however, provided unique opportunity to investigate the abnorm-a l i t i e s r e s u l t i n g from loss of LCAT a c t i v i t y . The f i r s t d e f i c i e n t patient was discovered i n June 1966 when a 33-year-old woman (A.R.) was admitted to the University Hospital i n Oslo. She had di f f u s e grayish corneal o p a c i t i e s , anemia, p r o t e i n u r i a and hypelipemia. However, her renal tubular function was normal and had no previous h i s t o r y of acute n e p h r i t i s . Further testing reviewed that both her plasma t r i g l y c e r i d e s and cholesterol were increased and most of the cho l e s t e r o l was u n e s t e r i f i e d . Her plasma l e c i t h i n was increased, plasma l y s o l e c i t h i n was decreased and no pre-beta or 0 t ^ - l i p O p r o t e i n could be detected on electrophoresis. Subsequent studies found that her two s i s t e r s (.M.R. and I.S.) expressed s i m i l a r c l i n i c a l features and the same plasma abnormalities. Later experimental studies disclosed the absence of plasma LCAT a c t i v i t y i n a l l three subjects. Since a l l three subjects had no previous h i s t o r y of l i v e r disease which could account for such abnormalities, i t appeared that they suffered from a previously undiscovered inborn error of - 14 -metabolism. Since then, twenty-one patients have been reported to be s u f f e r i n g from the disease (.36-42). Evidence accumulated so far favours the view that the disease is an expression of a homozygous autosomal recessive t r a i t , but whether d i f f e r e n t mutations are involved in production of what appears to be the same disease have not been determined. Patients with f a m i l i a l LCAT deficiency are c l i n i c a l l y characterized by d i f f u s e corneal opacity and arcus, normochromic anemia with decreased erythro-cyte l i f e span and most patients also suf f e r from proteinurea and develop renal i n s u f f i c i e n c y i n t h e i r fourth to f i f t h decade of l i f e . Premature atherosclerosis i s also an important feature of this disease. A l l of the c l i n i c a l features of the disease can presumably be explained by excessive l i p i d deposition i n the tissues due to abnormalities i n plasma li p o p r o t e i n s . Plasma lipoproteins of the patients are highly abnormal (Table I I I ) . They a l l contain a very low proportion of c h o l e s t e r y l esters and a high proportion of free cholesterol and l e c i t h i n (21,22,34,35). VLDL have a 8 mobility on electrophoresis (instead of pre-8) and contain a high concentration of very large p a r t i c l e s . The LDL portion of the plasma contains at least three groups of p a r t i c l e s (41,44,45): abnormally large p a r t i c l e s r i c h i n FC and l e c i t h i n , an intermediate-size group of p a r t i c l e s s i m i l a r to those found in obstructive jaundice (LP-X), and a f i n a l group of normal-size p a r t i c l e s containing about ten times the normal amount of TG (46,47). Very few normal HDL p a r t i c l e s can be found i n the patients' plasma. Instead, the HDL f r a c t i o n has both abnormally large and small p a r t i c l e s of unusual electrophoretic mobility and appearance under the electron-microscope. The predominant species consists of b i l a y e r disks (previously discussed) and the rest are apparently spherical structures containing mainly apo AI and phospholipids (48-50). Although no TABLE I I I . Major l i p i d s of plasma lipoproteins i n patients with f a m i l i a l LCAT defi c i e n c y * (umole/ml plasma) VLDL (d = 1.006 g/ml) Normal Patient M.R. Patient A.A. UC 0.084 0.223 2.060 CE 0.066 0.101 0.381 PC 0.077 0.186 1.210 TG 0.220 0.331 4.680 CE/UC 0.79 0.43 0.18 LDL 2 U = 1.019-1.063 g/ml) Normal 0.627 1.369 0.336 0.071 2.18 Patient M.R. 1.730 0.164 1.210 0.261 0.09 Patient A.A. 1.760 0.025 0.910 0.366 0.01 HDL (d = 1.063-121 g/ml) Normal Patient M.R. Patient A.A. 0.514 0.748 0.506 1.460 0.053 0.029 1.070 0.661 0.397 0.052 0.078 0.690 2.84 0.07 0.06 Abbreviations: UC = un e s t e r i f i e d c h o l e s t e r o l CE = chol e s t e r o l ester PC = phosphatidylcholine TG = t r i g l y c e r i d e *Data obtained from Reference 39 - 16 -q u a l i t a t i v e abnormalities can be seen, higher content of apo C-I and apo E can be found with a concurrent decrease i n apo A-I, apo C-II, C-III, apo B and apo D content (.51). The abnormal amounts of apoproteins may therefore contribute to the unusual electrophoretic mobility of the lipopr o t e i n s . Studies of erythrocyte and other peripheral c e l l membrane l i p i d composition i n LCAT deficiency also support Glomset's hypothesis that LCAT i s of importance i n the homeostasis of free cholesterol i n plasma membranes of peripheral t i s s u e . Both FC and l e c i t h i n contents are high i n the patients' erythrocytes and these abnormalities are reversed when erythrocytes are incubated with normal human plasma (52). These findings support the idea that LCAT i s at least i n d i r e c t l y involved i n the control of l i p i d on c e l l surface membrane and that these l i p i d s can e q u i l i b r a t e with lipo p r o t e i n s . As a r e s u l t a net removal of free cholesterol from peripheral c e l l to the plasma and i t s subsequent removal by the l i v e r i s possible. Many patients with l i v e r disease who have no, or a minimal, LCAT a c t i v i t y manifest l i p o p r o t e i n abnormalities s i m i l a r to those found in f a m i l i a l LCAT deficiency (53-55). Plasma FC and l e c i t h i n levels are elevated and abnormal VLDL and LDL molecules are also present i n these patients. The changes i n th e i r erythrocyte l i p i d composition also resemble those reported i n LCAT d e f i c i e n t patients (54,55). Of interest is the fact that patients with l i v e r disease who have normal LCAT a c t i v i t y u s u a l l y have none of the lip o p r o t e i n abnormalities described above (.54). These findings further confirm the important role of LCAT in l i p o p r o t e i n metabolism. Methods of LCAT assay Since the LCAT reaction provides most of plasma c h o l e s t e r y l esters and plays an important role i n l i p o p r o t e i n metabolism, a good understanding of the - 17 -factors c o n t r o l l i n g the secretion and a c t i v i t y of the enzyme would be valuable. However, l i t t l e is known of the factors that influence LCAT production and i t s secretion into plasma. One of the major problems i n i n v e s t i g a t i o n of the enzyme is the lack of a c l e a r i n t e r p r e t a t i o n of a c t i v i t y measurements and the absence of a s u i t a b l e standardized assay for plasma LCAT a c t i v i t y . Currently, levels of LCAT i n plasma are determined by functional assays in which the enzyme's a b i l i t y to e s t e r i f y cholesterol i s measured. There are three methods used to determine the rate of c h o l e s t e r o l e s t e r i f i c a t i o n . The f i r s t method was developed by Glomset and Wright C 57 i i n 1963. They added the sample serum to heat-inactivated plasma la b e l l e d with free J H - c h o l e s t e r o l and incubated this mixture at 37° for 1-8 hours. Changes i n the l a b e l l e d free cholesterol l e v e l would then be measured and expressed as the rate of cholesterol e s t e r i f i -cation. The disadvantage of this assay is that the use of heat-inactivated plasma can lead to an erroneous estimation of the LCAT a c t i v i t y since the rate of c h o l e s t e r o l e s t e r i f i c a t i o n in vivo depends on both enzyme a c t i v i t y and the amount (and q u a l i t y ) of c i r c u l a t i n g l i p o p r o t e i n s . The v a r i a t i o n in l i p o -protein composition of substrates that are prepared at d i f f e r e n t times (even from same subject) can be high and thus re s u l t s from d i f f e r e n t experiments may not be comparable. This method i s therefore not s u i t a b l e for c l i n i c a l i n v e s t i g a t i o n s . The second approach, developed by Stokke and Norum (1971), involves the pre-incubation of serum sample with la b e l l e d c h o l e s t e r o l in the presence of a d i s u l f i d e bond i n h i b i t o r (58). LCAT a c t i v i t y of the measured specimen is temporarily i n h i b i t e d by d i t h i o b i s - n i t r o b e n z o i c acid (DTNB) (usually one-half hour). During the i n h i b i t i o n the added l a b e l l e d cholesterol e q u i l i b r a t e s with that of endogenous li p o p r o t e i n s . F i n a l l y the enzyme is reactivated by addition of a t h i o l reagent and ester i f i c a t i o n a c t i v i t y is - 18 -followed for 30 minutes. This method i s currently widely used and i t seems to y i e l d comparable normal values (60-120 nmol/ml/hr) among d i f f e r e n t labor-a t o r i e s . However, the completeness of e q u i l i b r a t i o n between l a b e l l e d free c h o l e s t e r o l and l i p o p r o t e i n c h o l e s t e r o l is the most c r u c i a l step in the experiment and i t s t i l l remains to be shown whether this e q u i l i b r a t i o n procedure is applicable in the LCAT assay of hyperlipidemic sera. In the t h i r d method cholesterol ester i f i c a t i o n i s measured simply as a difference between concentration of FC at zero time and a f t e r the incubation at 37° for 30-60 minutes. This procedure (using g a s - l i q u i d chromatography) for estimation of FC i s very s e n s i t i v e and precise. However, the method is laborious and requires s p e c i a l equipment (59,60). Results from d i f f e r e n t runs also show large v a r i a t i o n s (58) and this technique cannot be used when the rate of c h o l e s t e r y l esters formation i s low or when small samples are to be used. Recent development of an a r t i f i c i a l substrate method (71), i n which sonicated l e c i t h i n / c h o l e s t e r o l emulsions are used together with apo-AI may prove to be the best a l t e r n a t i v e . This method does allow the preparation of a standardized substrate. However, no data on r e p r o d u c i b i l i t y of assays with the a r t i f i c i a l substrate are a v a i l a b l e . Moreover, the use of sample serum in the system is a disadvantage since the l i p o p r o t e i n l i p i d s i n the serum may e q u i l i b r a t e with the a r t i f i c i a l substrate and lead to inaccurate measurement of LCAT a c t i v i t y . Another approach to the assessment of LCAT is an immunoassay i n which the amount of LCAT protein rather than i t s e s t e r i f i c a t i o n a c t i v i t y is measured. This method of quantitating the enzyme as compared to the functional assay would have numerous advantages. Immunoassay not only allows a true assessment of LCAT protein l e v e l i n plasma, but also eliminates the problem of v a r i a b i l i t y i n substrates and the e f f e c t of i n h i b i t o r s and a c t i v a t o r s . Furthermore, the - 19 -importance of LCAT being complexed with other molecules (eg. HDL or apo D) (62) can be assessed when both the concentration and the enzymatic a c t i v i t y are measured simultaneously. This a p p l i c a t i o n is e s p e c i a l l y u seful i n comparing normal and diseased state. Study of l o c a l i z a t i o n and regulation of LCAT synthesis would thus also be f e a s i b l e . Development of a good immunoassay, whether i t is a r a d i o - or e l e c t r o -immunoassay, hinges on the production of pure enzyme (for l a b e l l i n g and standardization) and of a monospecific antibody against LCAT. A v a i l a b i l i t y of an adequate amount of pure enzyme and good a n t i g e n i c i t y of the preparation are e s s e n t i a l for the production of the antibody. F i n a l l y , the antibody has to be appropriately characterized. The p o t e n t i a l usefulness of such anti-LCAT antibody includes determination of the presence or absence of the LCAT enzyme in f a m i l i a l LCAT deficiency or i n other diseased states (eg. jaundice), the tes t i n g for c r o s s - r e a c t i v i t y with other species and the development of a f f i n i t y chromatography for the p u r i f i c a t i o n of LCAT. Additional information on the enzyme-lipoprotein complexes could also be gained (62). This thesis deals with the preparation of an antibody against LCAT and subsequent development of an immunoassay to quantitate the enzyme protein in plasma. Previously only two groups of researchers (63,64) were successful i n their attempts to raise antibodies against the enzyme. However they did not p e r s i s t further i n the development of an immunoassay. Furthermore, the data presented by these investigators were at least p a r t l y contradictory. One noticeable d i f f i c u l t y of this project was the preparation of the pure enzyme. Present method of i s o l a t i n g the enzyme are time consuming and the y i e l d is low. On the other hand, the limited success of the two previous groups of researchers showed that the project was f e a s i b l e . - 20 -MATERIALS AND METHODS Antigen preparation Enzyme p u r i f i c a t i o n Human LCAT was p u r i f i e d up to 15,000-fold by a modified Chung and Scanu procedure (65). The method combines the use of u l t r a c e n t r i f u g a t i o n and chromatographic techniques. The f i n a l preparation showed a single homogeneous protein band on a 7% polyacrylamide g e l . The enzyme has a molecular weight of 67-69,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzymatic a c t i v i t y was measured by a modified a r t i f i c i a l substrate assaying technique developed by Scanu et a l . (65). A more detailed summary of the p u r i f i c a t i o n procedure i s described below. Human plasma: Outdated human plasma (2 weeks old) i n 0.02M sodium c i t r a t e / 0 . 5 % dextrose was obtained from the Blood Bank of the Vancouver General Hospital, Vancouver, B.C. The plasma was centrifuged at 2,000 x g at 4°C for 30 minutes i n a Beckman J2-21 centrifuge to remove any sediment that might be present. The enzymatic a c t i v i t y was determined and recorded in plasma units (% FC e s t e r i f i e d / 2 0 y l s t a r t i n g plasma/4 h r ) . U l t r a c e n t r i f u g a t i o n : The plasma was adjusted to a s a l t density of 1.25 g/ml with s o l i d NaBr (Sigma Chemical Co., St. Louis, MO. 63178, USA) according to the formula described by Hatch and Lees (86) and centrifuged at 55,000 rpm for 20 hr i n a T i 60 (Beckman Ins., Palo A l t o , CA 94304; rotor at 10°C. The top 5 ml of the solution from each tube a f t e r c e n t r i f u g a t i o n were c o l l e c t e d and pooled together. The density was adjusted to 1.21 g/ml with addition of d i s t i l l e d H 2 o and centrifuged at 42,000 rpm in a T i 70 rotor (Beckman Ins.) for 64 hr at 10°C. The top 1 ml of the so l u t i o n a f t e r c entrifugation was - 21 -removed by tube s l i c i n g and about 4 ml of the middle fr a c t i o n s were c o l l e c t e d . This f r a c t i o n contained most of the LCAT a c t i v i t y (see Table IV). DEAE-Sephacel chromatography: The dialysed solution was applied to a Pharmacia column (2.6x30 cm) packed with DEAE Sephacel (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) e q u i l i b r a t e d at pH 7.4 with 1 mM T r i s , 5mM EDTA, 25 mM NaCl buffer at 4°C. The enzyme was eluted using a li n e a r NaCl gradient between 25 mM NaCl, 1 mM T r i s buffer, pH 7.4 and 5 mM NaCl, 10 mM T r i s buffer, 5mM EDTA, pH 7.4. Ten ml fractions were c o l l e c t e d as the column was eluted by pumping the buffer through at a flow rate of 48 ml/hr. Protein concentrations of the fracti o n s were monitored by measuring the absorbance at 280 nm in a Hita c h i Perkin-Elmer double beam spectrophotometer. E s t e r i f y i n g a c t i v i t y of the fract i o n s was also measured. Fractions which showed enzymatic a c t i v i t y were pooled together. A t y p i c a l DEAE-Sephacel chromatography p r o f i l e is shown in F i g . 5. The pooled fractions were concentrated by vacuum d i a l y s i s to about 1/20 of the o r i g i n a l volume and dialysed against several changes of 50 volumes of 5 mM sodium phosphate, 1 mM d i t h i o t h r e i t o l (DTT), pH 6.9. Hydroxylapatite chromatography: The hydroxylapatite column (1.6x26 cm) (Bio Gel HP obtained from Biorad Laboratory, Richmond, CA. USA 94804) chromato-graphy was the f i n a l step i n the p u r i f i c a t i o n scheme. The dialysed solution obtained a f t e r the DEAE-chromatography was applied onto the column and was eluted by pumping at a flow rate of 7 ml/hr with 5 mM sodium phosphate, 1 mM DTT, pH 6.9 at 4°C. Five ml fracti o n s were col l e c t e d and the LCAT enzyme was eluted as a single peak i d e n t i f i e d by the a c t i v i t y assay and the A o o r i of z o U a l l the f r a c t i o n s . A t y p i c a l p r o f i l e of the hydroxyapatite column is shown i n F i g . 6. - 22 -Pooled hydroxylapatite fractions were then concentrated by vacuum d i a l y s i s to about 100 Ug protein/ml. Purity of the LCAT enzyme was assessed by analysing the protein on a preparative polyacrylamide-gel electrophoretic system (Davis, 1964) (67). Immunization: New Zealand white rabbits weighing approximately 2.5 kg obtained from the Animal Care Unit, UBC were used as hosts for the antibody production. Before immunization, 10-20 ml of blood were co l l e c t e d from the ear veins of the r a b b i t s . The blood was allowed to c l o t and r e t r a c t and l a t e r centrifuged. Serum thus c o l l e c t e d was lab e l l e d as "pre-immunized (control) serum". For i n i t i a l i n j e c t i o n s the p u r i f i e d LCAT preparation was adjusted to a concentration of 1 mg protein/ml i n a 5 mM phosphate buffer, pH 6.9. One ml of enzyme solution was emulsified with an equal volume of complete Freund's adjuvant (DIFCO Lab., Detroit, MI. USA). Freund's complete adjuvant i s a mineral water-oil emulsion containing h e a t - k i l l e d human-type tubercle b a c i l l i . When mixed properly with the antigen the adjuvant can induce better immune response (68). Antigen was emulsified by repeated i n j e c t i o n s of the preparation into adjuvant through a #25 gauge needle u n t i l the mixture became thick and pasty. The emulsified enzyme was injected subcutaneously into the back of the rabbits at multiple s i t e i n j e c t i o n s . Fourteen to twenty days a f t e r the i n i t i a l i n j e c t i o n s , rabbits were bled again from the ear veins and serum was c o l l e c t e d to test for presence of antibody against LCAT. Regardless of the presence or absence of antibody, a booster shot was given to each animal. The enzyme preparation for the booster had a concentration of about 200-250 pg protein/ml and was mixed with i n -complete Freund's adjuvant. Seven to ten days a f t e r the last i n j e c t i o n 5-10 - 23 -ml of serum was c o l l e c t e d again for further c h a r a c t e r i z a t i o n . This cycle of boosting and bleeding was continued u n t i l no further increase i n antiserum t i t r e could be detected. Three rabbits were immunized with the p u r i f i e d LCAT preparation. A fourth rabbit was treated with Freund's adjuvant to serve as another control (.in addition to the pre-immunized serum). P u r i f i c a t i o n of gamma glob u l i n Antibody a c t i v i t y i n serum is largely associated with the gamma-globulin f r a c t i o n and p u r i f i c a t i o n of th i s f r a c t i o n i s f a i r l y simple (68). To every volume of rabbit serum c o l l e c t e d , a half-volume of saturated ammonium sulphate sol u t i o n was added with constant s t i r r i n g at 4°C. The mixture had a f i n a l concentration of 33% ammonium sulphate, and was allowed to stand i n 4°C for 15-30 minutes followed by cen t r i f u g a t i o n at 2000 rpm. The sediment was washed 3-4 times with 40% ammonium sulphate sol u t i o n , and dissolved i n a 0.9% saline s o l u t i o n . The solution was made up to the o r i g i n a l serum volume with normal saline s o l u t i o n , r e p r e c i p i t a t e d with a half-volume of saturated ammonium sulphate and washed using the same scheme as before. This procedure was repeated 3 times and the f i n a l sediment was dissolved i n 0.2 M sodium phosphate buffer pH 7.1 and dialysed exhaustively against the same buffer at 0°-4°C i n order to remove a l l the ammonium sulphate. The dialysed solution was concentrated to 1/3 of the o r i g i n a l serum volume by vacuum d i a l y s i s . Characterization of antibody Immunodiffusion: Ouchterlony double d i f f u s i o n technique (69) was used to evaluate the presence of a p r e c i p i t a t i n g antibody. Immunodiffusion plates were eit h e r purchased from Hyland (Div. Travenol Lab. Inc., Costa Mesa, CA) or prepared i n our own laboratory. Five ml of 1% agarose (Baker Grade, J.T. Baker Chem. Co., P h i l l i p s b u r g , NJ) was poured onto 25mm x 75mm microslides and - 24 -wells punched 4 mm apart. Antiserum were placed i n the middle well and the outer wells were f i l l e d with varying concentrations of antigens. The s l i d e was put i n a humid chamber and was incubated for 24-48 hr at room temperature. Immunoprecipitation li n e s could often be seen before s t a i n i n g but a l l s l i d e s were stained as described below. Staining and destaining: A f t e r the incubation, s l i d e s were pressed and washed for 2-3 hr with 0.9% NaCI solution to remove any non-precipitated proteins. This procedure was repeated twice and the gel was washed once more in d i s t i l l e d water, pressed and dried. The gel, appearing as a thin layer over the micro- s l i d e , was then put into a 0.5% Coomassie B r i l l i a n t Blue s t a i n i n g s o l u t i o n . The staining solution was prepared by d i s s o l v i n g Coomassie B r i l l i a n t Blue R-250 (Biorad Lab, Richmond, CA) i n e t h a n o l / g l a c i a l acetic a c i d / d i s t i l l e d H 20 (5:1:5). A f t e r approximately 20 minutes of staining the s l i d e was destained u n t i l c l e a r i n a destaining solution of ethanol/acetic a c i d / d i s t i l l e d H 2 o (5:1:5). Functional assays Stokke-Norum assay (70): A modified Stokke-Norum assay (58) described by Lacko et a l . (70) was used to measure the i n h i b i t o r y e f f e c t of the rabbit antibody on LCAT a c t i v i t y of normal human plasma. Unless otherwise s p e c i f i e d , 200 \il of f a s t i n g human plasma were incubated with various amounts of a n t i -serum or 'Y-glolmli - r i (both control and immunized). The f i n a l volume of the mixture was adjusted to 500 p i with 0.2 M sodium phosphate buffer pH 7.2. One hundred m i c r o l i t r e s of 4.2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (Sigma Chemical Co, St. Louis, MO) was added followed by an incubation period of 30 minutes at 37°C. The addition of DTNB r e v e r s i b l y i n h i b i t s the LCAT enzyme a c t i v i t y i n the normal human serum. A volume of 0.15 ml of 5% - 25 -bovine albumin s o l u t i o n (.Miles Lab. Inc, USA) containing 0.6 yCi 7- H cholesterol (NEN, Boston, Mass; 98% radiochemically pure) was added next and was allowed to e q u i l i b r a t e with endogenous lipoproteins i n the serum for 4 hr at 37°C. The c h o l e s t e r o l - e s t e r i f y i n g a c t i v i t y was l a t e r restored by the addition of 0.1 ml of 1 M 2-mercaptoethanol (Sigma Chem. Co.) and the reaction was stopped by removing 100 u l of the aliquots into 2 ml chloroform:methanol (2:1) at i n t e r v a l s (0, 10, 20, 40, 60 minutes). Extraction of l i p i d s by the chloroform/methanol mixture was continued overnight at room temperature and the extract was then dried down under a stream of nitrogen gas. The dried l i p i d residue was dissolved i n a small volume of chloroform and subjected to thin layer chromatography on thin layer plates of s i l i c a gel G (Brinkmann Instruments Inc., Westbury, N.Y. 11590). The plates were developed i n a solvent system of petroleum ether:ethyl e t h e r : g l a c i a l acetic acid (75:10:1, v/v), and l a t e r exposed to iodine vapour for v i s u a l i z a t i o n of l i p i d s . Free and e s t e r i f i e d c h o l e s t e r o l were i d e n t i f i e d , cut out and counted for radio-a c t i v i t y in a Beckman LS-9000 s c i n t i l l a t i o n counter. LCAT a c t i v i t y was expressed as % cholesterol e s t e r i f i e d / t i m e according to the following equation: dpm EC _ dpm EC  dpm FC + dpm EC t i m e a dpm FC + dpm EC t i m e Q\ x 100% A r t i f i c i a l substrate method I n h i b i t i o n of LCAT a c t i v i t y by antibody was also studied using an a r t i f i c i a l substrate of liposome v e s i c l e s of l e c i t h i n and free cholesterol (65). To prepare 1 ml of such v e s i c l e s , 0.788 mg of egg yolk l e c i t h i n and 96 pg of free cholesterol (Sigma chem. Co., St. Louis, MO) were mixed with 2 (iCi (2 ul) of 7-N- H free c h o l e s t e r o l (NEN) and dried under N^ gas. The dried mixture was redissolved i n 50 \il of absolute ethanol and rapidly injected into 1.0 ml of 40 mM sodium phosphate buffer, pH 7.2 through a Hamilton syringe to - 26 -form liposomal v e s i c l e s . This preparation was dialysed against 40 mM sodium phosphate to remove ethanol. A f t e r d i a l y s i s the v e s i c l e s were ready to be used for assaying LCAT a c t i v i t y . F i f t y m i c r o l i t r e s of the dialysed v e s i c l e s were used per assay and they were incubated with 50 y l of apo A-I protein (12.5 yg) (prepared i n our laboratory (17,24)) for 30 minutes at 37°C under nitrogen. F i n a l l y , 50 y l of pH 7.2 buffer containing 40 mmoles of sodium phosphate, 4 mmoles EDTA, 50 g human serum albumin ( f a t t y acid free, Mann Research Lab, Inc., N.Y.J and 16 mmole B-mercaptoethanol (Sigma) per l i t r e were added to the mixture to complete the substrate preparation. Twenty to twenty-five m i c r o l i t r e s of the p u r i f i e d (see above) enzyme were mixed with various amounts of antiserum or gamma gl o b u l i n ( e i t h e r pre- or post-immunized). The f i n a l volume of the assay mixture was made up to 200 y l with the sodium phosphate buffer and the solution was usually incubated for 15-20 minutes. The reaction was stopped and i s o l a t i o n of free and e s t e r i f i e d c h olesterol were done with the same procedure as described for the Stokke-Norum assay. Immunoassay: Rocket Immunoelectrophoresis (72;. This i s a quick and simple method for i d e n t i f i c a t i o n and quantitation of a s p e c i f i c protein i n a protein mixture such as plasma. During electrophoresis i n agarose gel saturated with antibody, the antigen interacts with the antibody to form a rocket-shaped p r e c i p i t a t e . Quantitation is based upon the height or area of the p r e c i p i t a t e ("rocket"). One ml of antiserum was mixed with 9 ml of 1% agarose sol u t i o n (50-60°C) and poured onto a 8x10 cm heated glass p l a t e . The gel plate was allowed to cool and 2 mm diameter wells were punched side by side along one side of the - 27 -g e l . The gel was leveled on the electrophoresis apparatus and 10 p i samples were pipetted into the wells. Gels were then connected to the buffer chambers through wicks and approximately 1.5 l i t r e s of barbital-Na/ b a r b i t a l buffer with pH 8.8 and ioni c strength 0.02 was poured into the chambers. E l e c t r o -phoresis was conducted overnight at 10-12°C at 70-100 V. The gels were washed and stained as described previously for immunodiffusion. Protein was quantitated by measuring the area of the "rockets". Measure-ment of the peak height and width of the p r e c i p i t a t e s was c a r r i e d out on graph paper marked with one millimetre squares with the gel side of the plate facing the paper. Standard curves were drawn by p l o t t i n g area against known concen-t r a t i o n of standard samples. The r e l a t i o n s h i p between concentration of protein and the area of the p r e c i p i t a t e was l i n e a r . The i n t e n s i t y of the p r e c i p i t a t e was enhanced by varying the proportions of antigen and antibody used. For example when the p r e c i p i t a t e s are too f a i n t , more antiserum should be used. A number of factors are known to influence the height of the p r e c i p i t a t e s and therefore i t was f e l t important to keep these factors as constant as possible for reproducible r e s u l t s . These factors include (72): 1. antigen concentrations 2. antibody concentration 3. f i e l d strength of electrophoresis (V/cm) 4. buffer pH and ioni c strength 5. gel concentration 6. duration of electrophoresis Protein determination (73) Lowry protein assay (74) as modified by Peterson (73) was used. This procedure allowed for analysis of very d i l u t e protein solutions plus the - 28 -removal of many unwanted i n t e r f e r i n g substances that may be present. Deoxycholate-tricholoracetic acid (DOC-TCA) was used to achieve quantitative p r e c i p i t a t i o n of minute amounts of protein (75) and the addition of sodium dodecyl sulfate f a c i l i t a t e d the removal of i n t e r f e r i n g substances. A general scheme of the procedure i s given below: 1. To 1 ml of protein s o l u t i o n , either standards or unknown samples, 100 p i of 0.15% deoxycholate solution was added (ICN Pharmaceuticals Inc., Cleve. Ohio) and the mixture allowed to stand for 10 minutes. 2. 100 y l of 72% (w/v) t r i c h o l o r o a c e t i c acid was added (Fischer S c i e n t i f i c Co., NJ) followed by c e n t r i f u g a t i o n at 6500 rpm for 15 minutes. 3. Supernatant was discarded and 1 ml of d i s t i l l e d r^O was added to the p e l l e t . 4. 1 ml of reagent A was then added and the so l u t i o n was well mixed and allowed to stand for 10 minutes at room temperature. 5. Reagent B (0.5 ml) was put i n and absorbance was read at 750 nm a f t e r 30 minutes. Reagent A: mix equal parts of Copper-tartrate-carbonate (CTC), 10% (w/v) sodium dodecyl sulphate (SDS.). 0.8 N sodium hydroxide (Fischer S c i e n t i f i c Co., NJ) and d i s t i l l e d H 2Q. Reagent B: Folin-Ciocalteau phenol reagent (Fischer S c i e n t i f i c Co., NJ) d i l u t e d 1:6 with d i s t i l l e d H2o. A s t r a i g h t l i n e was obtained when the log of the absorbance was plotted against the log of protein concentration of the standards. Bovine albumin (Miles Lab, Inc., USA) of concentrations ranging between 25 ug/ml to 100 ug/ml were used as standards. - 29 -RESULTS Antigen Preparation T y p i c a l DEAE-sephacel and hydroxylapatite chromatography p r o f i l e s are shown in F i g . 5 and 6 respectively. Protein electrophoresis of the p u r i f i e d enzyme preparation on a 7% polyacrylamide gel showed a single homogeneous protein band ( F i g . 7). Gel was s l i c e d into 2mm sections and LCAT a c t i v i t y was measured. Enzymatic a c t i v i t y was only found i n s l i c e s corresponding to the stained band. Characterization of rabbit anti-LCAT serum A) Immunodiffusion Formation of immunoprecipitate was detected between p u r i f i e d LCAT enzyme and rabbit antiserum obtained a f t e r ten weeks of immunizations. Se r o l o g i c a l i d e n t i t y was also observed between human plasma and the p u r i f i e d enzyme preparation whereas no l i n e of p r e c i p i t a t i o n was formed when LCAT d e f i c i e n t plasma was tested against the antiserum. The use of r a d i a l immunodiffusion technique enabled one to see the differ e n c e c l e a r l y ( Fig. 8b;. In this s i t u a t i o n the antiserum is mixed with the agarose sol u t i o n , and the antigen d i f f u s e s into the gel to react with the antiserum. Any r e s u l t i n g p r e c i p i t a t i o n would be in the form of a ri n g surrounding the well. Figure 8b shows that only the well containing the normal human plasma gives a p o s i t i v e reaction. This finding is i n agreement with those reported by Utterman et a l . (33), and suggests that f a m i l i a l LCAT-deficient patients do not have immunoreactive LCAT in t h e i r plasma. F i g . 8a also shows that the anti-LCAT serum did not react with human serum albumin (60 mg/ml) and apo A-I protein (250 yg/ml). When isola t e d l i p o p r o t e i n fractions (84) were tested against the antiserum, only - 30 -TABLE IV. P u r i f i c a t i o n of l e c i t h i n : c h o l e s t e r o l acyltransferase Volume Total Total ml protein P.U. (mg) Sp. Act. Y i e l d P u r i f i -PU/mg (%) cation Plasma 610 d = 1.25 g/ml 475 bottom d = 1.21 g/ml 92 middle d = 1.21 g/ml 27.5 bottom DEAE 158 pool 38740 19140 555.5 610 100.5 237.4 260.4 29.07 164.3 0.016 100 1 0.010 32.7 1.097 20.63 0.037 5.652 42.7 69.7 3.4 26.9 359 HA pool 38 0.199 26.98 226.7 4.4 14394 P.U. = Plasma u n i t : % free c h o l e s t e r o l e s t e r i f i e d / 4 hr/20 y l plasma - 31 -2.0 § 1 - 0 CM < i i X nth \ J / \ t * » x-'- 4 L - t -1" 4 rf E J , c o u i i i •15 r - 1 0 20 40 60 •< o > CM u D *»• Q r— f-< CO 111 u 1 N9 •3.0 •2.0 •1.0 80 100 FRACTION DEAE-Sephacel column chromatography of the dialysed d = 1.21-1.25 g/ml f r a c t i o n . Enzymatic a c t i v i t y was measured by an a r t i f i c i a l substrate system as described by Scanu et a l . (65). For d e t a i l s see 'Materials and Methods'. \ - 32 -FRACTION F i g . 6. Hydroxylapatite column chromatography of the active DEAE-Sephacel eluates. The LCAT enzyme was eluted with 5 mM sodium phosphate 1 mM DTT pH 6.9 buffer as a s i n g l e peak. - 33 -F i g . 7. P u r i f i e d LCAT (30lig) electrophoresed on 7% polyacrylamide gel according to Davis (1964). Gel was satined with 0.5% Coomassie B r i l l i a n t Blue R-250 solution. - 34 -F i g . 8a. Immunodiffusion of anti-LCAT antiserum against human plasma and pu r i f i e d LCAT preparation. HP = human plasma, DH = d e f i c i e n t plasma, HSA = human serum albumin, HHP = heated human plasma, VLDL = very high density l i p o p r o t e i n , LDL = low density l i p o p r o t e i n , HDL = high density l i p o p r o t e i n , VHDL = very high density l i p o p r o t e i n (d 1.21-1.25 g/ml). Anti-LCAT serum CIO was put i n the centre well and 10 Ml of each sample to be tested were applied to the outside wells. P o s i t i v e r e a c t i v i t i e s can be seen between antiserum and p u r i f i e d LCAT, normal human plasma and the isol a t e d VHDL f r a c t i o n . F i g . 8b. Radial immunodiffusion. Anti-LCAT serum (250 was mixed with 3 ml of 1% agarose solution and the mixture was poured onto a microslide. Left = DH, middle = normal plasma, r i g h t = SF SF and DH are LCAT d e f i c i e n t patients. - 36 -the d = 1.21-1.25 g/ml f r a c t i o n shows immuno- p r e c i p i t a t i o n . This r e s u l t agrees with the fact that most of the LCAT a c t i v i t y is found i n this f r a c t i o n (.see p u r i f i c a t i o n p r o f i l e ) . Isolated y - g l o b u l i n fractions from the antiserum also reacted p o s i t i v e l y with human plasma and the p u r i f i e d enzyme. The t i t r e of the antiserum was established by testing varying amounts of the p u r i f i e d LCAT preparation through double immunodiffusion and i t was found that 10 y l of the antiserum was sen s i t i v e enough to detect a 10 y l volume of 50 mg/1 of enzyme. B) Immunoinhibition of LCAT a c t i v i t y The a b i l i t y of anti-LCAT antibody to i n h i b i t LCAT a c t i v i t y as measured by functional assay was an important c r i t e r i o n i n assessing the t i t r e of the antibody produced. The i n h i b i t o r y e f f e c t of the antiserum was investigated in two d i f f e r e n t assay systems. The modified Stokke-Norum assay was used to measure the antiserum's a b i l i t y to i n h i b i t plasma LCAT a c t i v i t y whereas the a r t i f i c i a l substrate assay was employed to study the e f f e c t of the anti-LCAT serum on p u r i f i e d LCAT enzymatic a c t i v i t y . Antisera that were used in these functional assays studies were a l l pre-heated at 56°C for 30 minutes to eliminate any i n t e r f e r r i n g cholesterol e s t e r i f y i n g a c t i v i t y that may be present. I. I n h i b i t i o n of plasma LCAT a c t i v i t y i ) Plasma LCAT a c t i v i t y was measured according to the assay developed by Lacko et a l . (70) as described i n the Methods. The influence of the antiserum on normal plasma LCAT a c t i v i t y was measured by a decrease in e s t e r i f i c a t i o n rate and expressed as a percentage i n h i b i t i o n of the LCAT a c t i v i t y as compared to the normal co n t r o l values. The i n h i b i t i o n p r o f i l e is shown in F i g . 9 and i t is evident that progressive i n h i b i t i o n of human plasma LCAT a c t i v i t y - 37 -occurred when increasing amounts of the anti-serum were used. Complete i n h i b i t i o n of LCAT a c t i v i t y in 200 p i of plasma was achieved when 100 p i or more of the antiserum was used (Table V - l , V - l l ) . Figure 9 also shows that normal rabbit serum i n h i b i t e d LCAT a c t i v i t y by 20-30%. This non-specific i n h i b i t i o n was previously reported by Akanuma et a l . (80) in 1978. No appreciable difference i n the i n h i b i t o r y e f f e c t of the antibody was seen when iso l a t e d gamma-globulin f r a c t i o n was used instead of the antiserum. However, examination of the data indicates that the i n h i b i t o r y e f f e c t of the immunoglobulins was less concentration dependent. i i ) In order to determine the time course of i n h i b i t i o n , anti-LCAT serum was added to human plasma a f t e r the plasma had reached equilibrium with the labe l l e d free c h o l e s t e r o l (see Method). LCAT a c t i v i t y was measured at 5, 10, 15, 20, 30 and 40 minute i n t e r v a l s . The results are shown in Table VI. It i s obvious from the time course p r o f i l e ( F ig. 10a, 10b) that the degree of i n h i b i t i o n i s time-dependent and reaches the maximum at 40 minutes. However, when pre-immunized normal rabbit serum was used, a d i f f e r e n t p r o f i l e was seen. There was no s i g n i f i c a n t difference i n the degree of i n h i b i t i o n at d i f f e r e n t times caused by normal rabbit serum. This time-independent p r o f i l e i s c h a r a c t e r i s t i c of non-specific i n h i b i t i o n . D i f f e r e n t normal rabbit sera were used i n the experiments and a v a r i a t i o n was noted in t h e i r non-specific i n h i b i t i o n of LCAT a c t i v i t y (compare F i g s . 9 and 10b). i i i ) As previously shown by immunodiffusion, sera from the two patients with LCAT deficiency contained no immunoreactive LCAT. To further confirm this observation the functional assay was used. Pre-incubation of LCAT d e f i c i e n t plasma with the anti-LCAT serum should not r e s u l t i n an immuno-p r e c i p i t a t i o n due to absence of the enzyme. Nor should i t abolish the - 38 -TABLE V - l . INHIBITION OF NORMAL HUMAN PLASMA LCAT ACTIVITY 3 BY CONTROL RABBIT SERUM AND RABBIT ANTISERUM TO LCAT. Fresh human Post-immunized Pre-immunized Buffer % FC e s t e r i f i e d % i n h i b i t i o n plasma serum serum per hr ( y i ; ( yi) (yD ' (yL; + S . D + S . D 200 - - 200 4.67 + 0.65 -200 50 - 150 2.34 + 0.24 50 + 12 200 75 - 125 1.25 + 0.38 74 + 30 200 100 - 100 0.24 + 0.10 95 + 50 200 150 - 50 0.09 + 0.04 100 200 200 - - 0.06 + 0.04 100 200 - 50 150 4.03 + 0.14 14 + 3 200 - 100 100 3.68 + 0.07 22 + 3 200 - 125 75 3.85 + 0.10 18 + 3 200 - 200 - 3.38 + 0.17 28 + 7 aLCAT a c t i v i t y n = 12 measured by modified Stokke--No rum assay TABLE V-2. EFFECT OF IMMUNOGLOBULIN ON LCAT ACTIVITY Fresh human Post-immunized Pre-immunized Buffer % FC e s t e r i f i e d % i n h i b i t i o n plasma y-G y-G per hr (Ml) ( yl) ( p i ; (UL) + S.D + S.D. 200 50 - 150 0.66 + 0.28 86 + 47 200 100 - 100 0.81+0.16 83 + 16 200 150 - 50 0.43+0.03 9 0 + 1 9 200 200 - - 0.61+0.19 8 7 + 3 5 200 - 50 150 4.56+0.09 3+ 3 200 - 100 100 3.68 + 0.16 22 + 16 200 - 200 - 4.42+0.09 2 1 + 1 6 200 - - 200 4.67 + 0.60 n = 10 - 40 -post-immunized F i g . 9. Immunoinhibition of plasma LCAT a c t i v i t y . E f f e c t of various amount of anti-LCAT antibody on 200 u l of normal human plasma LCAT ac t i v i t y . n = 12 + S.E.M. - 41 -TABLE VI. TIME COURSE PROFILE Fresh human TIME Post-immunized Pre-immunized Buffer % FC % plasma serum serum e s t e r i f i e d i n h i b i t i o n (liU (min) (yl) ( y l ) y l + S.D. + S.D. 200 5 - - 200 0.26 + 0.03 -200 10 - - 200 0.94 + 0.08 -200 15 - - 200 1.41 + 0.08 -200 20 - - 200 1.71 + 0.06 -200 30 - - 200 2.92 + 0.08 -200 40 - - 200 3.49 T 0.14 -200 5 200 - - 0.16 + 0.03 38 + 8 200 10 200 - 0.25 + 0.03 74 + 17 200 15 200 - - 0.36 + 0.08 79 + 22 200 30 200 - - 0.42 0.08 86 + 22 200 40 200 - - 0.13 + 0.03 96 + 18 200 5 - 200 - 0.16 + 0.08 36 + 25 200 10 - 200 - 0.62 + 0.08 35 + 8 200 20 - 200 - 0.97 + 0.17 43 + 8 200 40 - 200 - 1.93 + 0.03 45 + 5 n = 8 - 42 -normal p lasma 10 2 0 30 4 0 Time (minutes F i g . 1 0 a . I n h i b i t i o n of plasma LCAT a c t i v i t y by anti-LCAT antiserum. Normal human plasma (200 ul.) was incubated with 200 p i of pre- or postimmunized rabbit serum. A c t i v i t y of the enzyme was measured at various times, n = 8 + S.E.M. - 43 -F i g . 10b. Percent i n h i b i t i o n of plasma LCAT a c t i v i t y by anti-LCAT antibody as a function of time, n = 8 _ + S.E.M. - 44 -antiserum's a b i l i t y to i n h i b i t LCAT a c t i v i t y since the antibody was not being removed. Heated normal human plasma was used as a control and the experiment was done according to the method previously described for the a r t i f i c i a l substrate assay system with some adjustments. D e f i c i e n t plasma Cor heated control plasma) was pre-incubated overnight at 40°C with anti-LCAT antiserum. The reaction mixtures were then centrifuged at 50,000 x g i n a Beckman a i r fuge and the top 100 u l of the so l u t i o n was removed and measured for anti-LCAT a c t i v i t y ( F ig. 11). Results are summarized in Table VII-1. The data reveal that s i g n i f i c a n t difference e x i s t s between the control plasma and the d e f i c i e n t plasma i n t h e i r a b i l i t y to a f f e c t the anti-LCAT serum's i n h i b i t o r y a c t i v i t y . Much of the antiserum's i n h i b i t o r y e f f e c t was abolished a f t e r the overnight incubation with the control plasma (% i n h i b i t i o n decreased from 95 to 70% and from 74 to 33%, F i g . 11). Some of the antibodies were removed through immunoprecipitation and as a r e s u l t less was available for i n h i b i t i o n in the subsequent assay. Yet when LCAT-deficient plasma was used, no such decrease i n the antiserum i n h i b i t o r y e f f e c t was observed. This i n d i r e c t l y supports the idea that normal LCAT enzyme does not exist i n plasma of patients with LCAT de f i c i e n c y . I I . I n h i b i t i o n of p u r i f i e d LCAT The a b i l i t y of the anti-LCAT serum to i n h i b i t the LCAT a c t i v i t y was also studied i n the a r t i f i c i a l substrate assay system. This assay has the advantage that only the p u r i f i e d enzyme plus a few other co-factors (apo-AI, human serum albumin) are used and thus a more d i r e c t and accurate measurement of the i n h i b i t o r y e f f e c t can be made. Isolated gamma glo b u l i n fractions were used instead of the whole serum i n order to eliminate any possible interference due to the presence of lipoproteins i n the rabbit serum. E f f e c t of incubation - 45 -TABLE VII-1 . EFFECT OF LCAT DEFICIENT PLASMA ON ANTI-LCAT SERUM Fresh human De f i c i e n t Heated normal Anti-LCAT Buffer X FC I plasma plasma plasma serum e s t e r i f i e d i n h i b i t i o n per hr (HU c y i ; (MU (MD Ml + S.D. + S .D. 100 - - - 100 6.72+0.33 -100 - - 50 100 0.35 + 0.22 95 + 49 100 - - 25 125 1.79 + 0.22 74 + 11 100 100 - 50 - 0.15 + 0.04 98 + 22 100 100 - 25 25 1.02 + 0.29 85 + 22 100 - 100 50 - 1.96 + 0.29 70 + 11 100 _ 100 25 4.51 + 0.31 33 + 4 See text for d e t a i l s n = 5 - 46 -antiserum + L CAT deficient plasma / i l of a n t i - L iCAT serum F i g . 11. E f f e c t of LCAT-deficient plasma on anti-LCAT a c t i v i t y of antiserum: Plasma from LCAT d e f i c i e n t patient (100 y l ) were pre-incubated with various amount of anti-LCAT antiserum overnight at 4°C. A f t e r c e n t r i f u g a t i o n , 100 y l aliquots were taken and measured for anti-LCAT a c t i v i t y . Heat-inactivated normal plasma were used as c o n t r o l , n = 5 + S.E.M. - 47 -time and antibody concentration on LCAT a c t i v i t y were again investigated. Results are shown in Figures 12 and 13. These two figures exhibit r e s u l t s very s i m i l a r to those presented e a r l i e r i n Figures 9 and 10. The antiserum does a f f e c t LCAT e s t e r i f y i n g a c t i v i t y ( i n plasma or p u r i f i e d form), and this antiserum appears to be directed against the enzyme LCAT. Immunoassay of human plasma LCAT 'Rocket' immunoelectrophoresis The plasma l e v e l of LCAT protein was measured by electroimmunodiffusion. Because of low t i t r e , one ml of anti-LCAT serum had to be used per assay i n order to achieve a p r e c i p i t a t e intensive enough to allow an accurate quantitation of protein. Standards were prepared from p u r i f i e d LCAT enzyme. Figure 14 shows that a l i n e a r r e l a t i o n s h i p exists between the area of the 'rockets' and the LCAT-protein concentration. The c o r r e l a t i o n c o e f f i c i e n t i s 0.99. The y-intercept r e f l e c t s the area of the wells. The plasma enzyme lev e l s for normal i n d i v i d u a l s were then determined, and LCAT protein concentration i n normal human plasma was found to be 5.40 + 0.54 mg/1 (.range: 4.49-5.94 mg/1). Because of the large amount of antiserum required per assay, the above value r e f l e c t s studies from only 12 normal i n d i v i d u a l s (samples done i n duplicate). When plasma from the two LCAT d e f i c i e n t patients was assayed no v i s i b l e p r e c i p i t a t e was formed. These findings confirm that these patients do not have any immunoreactive LCAT protein i n t h e i r plasma. The family members of the two patients were also studied (both parents and 4 s i b l i n g s ) . Their values are summarized in Table IX. Both the patients' parents and two of t h e i r s i b l i n g s have sub-normal quantities of enzyme, a finding compatible with autosomal recessive trans-mission of f a m i l i a l LCAT deficie n c y . A c o r r e l a t i o n between LCAT mass and i t s enzymatic a c t i v i t y i n these subjects was also investigated. The LCAT enzymatic a c t i v i t i e s were measured with the a r t i f i c i a l substrate method and data were compared to t h e i r respective enzyme mass. Results are l i s t e d i n Table X, and the s i g n i f i c a n c e of t h i s study w i l l be discussed l a t e r . - 49 -loo'r Time (minutes) F i g . 12. Anti-LCAT gamma-globulin (150 Mg) was added to 20 Ml of p u r i f i e d LCAT and the i n h i b i t o r y e f f e c t was followed for 40 minutes, n = 6 - 50 -163.75 100 200 300 400 500 y - g l o b u l i h ^tg (pre- or p o s t - i m m u n i z e d ) F i g . 13. Immune- i n h i b i t ion of p u r i f i e d LCAT a c t i v i t y i n an a r t i f i c i a l substrate system by anti-LCAT antibody. 20 ul of p u r i f i e d enzyme were used. • n = 10 E s t e r i f i c a t i o n a c t i v i t y in one ml of p u r i f i e d LCAT = 160 nmole FC ester i f i e d / h r . - 51 -0 1 2 3 4 5 6 Protein concentration of standard LCAT solutions (mg/l). F i g . 14. Standard curve for the determination of plasma protein l e v e l of LCAT. Pr o t e i n concentrations of LCAT solutions were determined by a modified Lowry procedure with bovine serum albumin as reference standards (73). The y-intercept r e f l e c t s the area of the wells. Mean + S.D. n = 9 - 52 -TABLE VIII-1. INHIBITION OF PURIFIED LCAT ENZYME3 LCAT Post-y-G Pre-y-G % FC est/15 min nmole FC est % inh ib i t ion (Ml) (Mg) (Mg) + S .D. ml LCAT hr 20 _ _ 6.25 + 0.15 163.75 _ 20 75 - 4.53 + 0.26 118.69 28 20 100 - 2.74 + 0.43 71.79 57 20 150 - 1.82 0.25 47.68 70 20 300 - - 100 20 450 - - - 100 20 - 75 4.40 + 0.30 115.28 30 20 - 100 4.21 + 0.28 110.30 33 20 - 150 4.17 + 0.17 109.25 37 20 - 300 3.75 7 0.30 98.25 40 20 450 3.80 + 0.27 99.56 50 3LCAT a c t i v i t y measured by a r t i f i c i a l substrate assay (13.1 nmole of FC per assay) n = 10 ml LCAT 160 nmole FC e s t e r i f i e d / h r - 53 -TABLE VIII-2. INHIBITION OF PURIFIED LCAT ENZYME AS A FUNCTION OF TIME nmole FC e s t e r i f i e d % FC e s t e r i f i e d + S.D. per ml LCAT % i n h i b i t i o n Time C o n t r o l s 3 with anti-LCAT b Controls with anti-LCAT (minute) Y-globulm % - g l o b u l i n 5 2.13 + 0.15 - 14.4 - -10 4.02 + 0.11 2.54 + 0.15 26 .2 16.4 47 20 8.23 + 0.18 2.66 + 0.24 53.4 17.0 68 30 10.75 + 0.26 2.45 + 0.16 72.0 16.1 78 40 12.25 + 0.20 2.74 + 0.15 79.8 18.0 78 a20 y l of p u r i f i e d LCAT were used per assay b150 yg of y-gl o b u l i n were added Addition of y-gl o b u l i n was made 5 minutes a f t e r the e s t e r i f i c a t i o n had been i n i t i a t e d . See Figure 12 for d e t a i l s . n = 6 - 54 -TABLE IX. QUANTITATION OF PLASMA LCAT BY 'ROCKET' IMMUNOELECTROPHORESIS Sample Area of rockets Concentration (cm 2) Mean S.D. (mg/1) Comments Standard 1 0 .28 0.03 5 .32 Standard solutions are Solution 2 0 .17 0.02 2 .66 p u r i f i e d 3 0 .15 0.01 1 .77 LCAT preparation 4 0 .12 0.01 1. .33 9 determinations were done on each sample Control plasma 0 .27 0.04 5.16 + 0.38 C.V.=7.4% (10 determin-ations) ran as q u a l i t y control for each assay Normal plasma 0 .28 0.04 5.40 + 0.54 12 donors (duplicate on each sample) Family members GF 0 .21 0.10 3.4 + 1.8 of d e f i c i e n t DF 0 .21 0.03 3.4 + 0.2 patient RF 0 .21 0.03 3.3 + 0.5 3 determinations were LF 0 .21 0.03 3.4 + 0.5 done on each sample AF 0 .27 0.04 5.1 + 0.5 DB 0 .31 0.04 6.1 0.5 SF -DH -GF and DF = parents RF and LF = brothers AF and DB = s i s t e r s SF and DH = LCAT d e f i c i e n t patients - 55 -F i g . 15. "Rocket" Immunoelectrophoresis of l e c i t h i n : c h o l e s t e r o l a c y l -transf erase. Wells 1-8 from l e f t : d i f f e r e n t d i l u t i o n s of the p u r i f i e d enzyme (1-6 mg/1). Well 9: normal control serum. Well 10: hyperlipemic serum. 1 ml of rabbit anti-LCAT antiserum was mixed i n with 9 ml of 1% agarose solution. 10 y l of sample was applied into each well. Electrophoresis was carried out at pH 8.6 using b a r b i t a l buffer with a f i e l d strength of 80V at 10-15°C overnight. For d e t a i l s see text. - 56 -F i g . 16. Quantitation of plasma LCAT in family members of LCAT d e f i c i e n t patients by electroimmunoassay. From l e f t to r i g h t : p u r i f i e d LCAT enzyme, p u r i f i e d LCAT + SF, SF, DH, GF, RF, DB, AF, LF, DF. GF, DF = parents DB, RF, LF, AF = s i b l i n g s - 57 -TABLE X. PLASMA LIPIDS AND LCAT VALUES IN FAMILY WITH LCAT DEFICIENCY Total c h o l e s t e r o l (mg/dl) Free cho l e s t e r o l U g/dl) Tr i g l y c e r i d e s (mg/dl) *LCAT a c t i v i t y nmole FC est./ ml plasma/hr LCAT conc'n (mg/1) Normal 130-250 39-78 52-180 45-85 4.2-5.8 GF 260 74 216 35.1 3.4 DF 199 58 167 25.9 3.4 AF 204 50 97 60.8 5.1 DB 248 63 233 61.6 6.1 RF 189 55 232 37 .3 3.3 LF 208 56 123 31.3 3.4 c o r r e l a t i o n c o e f f i c i e n t r = 0.93 n = 6 *LCAT a c t i v i t y measured by a r t i f i c i a l substrate assay (-71) Cholesterol and t r i g l y c e r i d e s were enzymatically determined on an Abbott Bichromatic Analyser (ABA-100) (78). - 58 -DISCUSSION This thesis deals with the development of an electroimmunoassay for human plasma enzyme l e c i t h i n : c h o l e s t e r o l acyltransferase. This immunoassay is simple and s p e c i f i c . It allows a d i r e c t quantitation of the enzyme protein i n plasma and provides an opportunity to study the re l a t i o n s h i p between the protein mass and the enzymatic a c t i v i t y . This comparison w i l l be valuable i n studies dealing with the control of LCAT reaction and permits a d i s t i n c t i o n between abnormalities i n the secretion of the enzyme and abnormalities involving substrate or cofactors. The v a l i d i t y of the immunoassay was established using several c r i t e r i a to v e r i f y i t s s p e c i f i c i t y for LCAT. F i r s t , the a n t i LCAT serum formed a sing l e and s e r o l o g i c a l l y i d e n t i c a l p r e c i p i t a t i o n arc against both p u r i f i e d enzyme and human plasma. Secondly, the antiserum d i d not react with any of VLDL, LDL, HDL, apo A-I or human serum albumin. Furthermore, the antiserum reacted strongly with the density 1.21-1.25 g/ml fractions of normal human sera which contained most of the LCAT enzymatic a c t i v i t y . T h i r d l y , incubation of the antiserum with e i t h e r human plasma or p u r i f i e d enzyme i n h i b i t e d the LCAT reaction completely. F i n a l l y , the antiserum f a i l e d to react with plasma of LCAT-deficient patients. Moreover, adsorption of the antiserum with normal human plasma led to a s i g n i f i c a n t decrease i n i t s anti-LCAT a c t i v i t y . In contrast, no such decrease was observed after, treatment of the antiserum with LCAT d e f i c i e n t plasma. The observation that s i g n i f i c a n t differences exist between pre- and post-immunized sera i n the i r i n h i b i t i o n of LCAT a c t i v i t y provides strong evidence that the i n h i b i t i o n was not the r e s u l t of any a r t i f a c t u a l e f f e c t due to the - 59 -addition of rabbit serum or gamma g l o b u l i n . The antibody showed a p o s i t i v e reaction with both human plasma and p u r i f i e d LCAT enzyme in immunodiffusion and hence the i n h i b i t i o n was probably due to binding of anti-LCAT antibody with enzyme which rendered the LCAT molecule inaccessible to the substrate - a prerequisite for cholesterol e s t e r i f i c a t i o n . Whether the binding between the antibody and the LCAT molecule occurred at the enzyme's active s i t e is not known. The a b i l i t y of the antiserum to i n h i b i t LCAT reaction i n both human plasma and i n p u r i f i e d enzyme preparation also favours the argument that the antibody i s s p e c i f i c a l l y directed against LCAT. Had the antibody been raised against some other contaminants, the antiserum should f a i l to i n h i b i t completely the p u r i f i e d enzyme a c t i v i t y when measured by the a r t i f i c i a l substrate method. Moreover, further addition of substrate to the reaction mixture f a i l e d to release the i n h i b i t o r y e f f e c t of the antibody had on the e s t e r i f i c a t i o n reaction. This i s evidence that the antibody was not directed against any of the substrate proteins. It should be emphasized that normal rabbit sera i n h i b i t LCAT reaction to varying degrees. This non-specific i n h i b i t i o n may well represent a decrease i n the a v a i l a b i l i t y of the radioactive substrate due to the addition of r a b b i t s ' lipoproteins contained i n the antiserum. The finding that the two subjects with f a m i l i a l LCAT deficiency have no detectable enzyme l e v e l i s i n good agreement with the results reported by Albers et a l . (76). Albers measured LCAT concentration by radioimmunoassay i n normolipidemic subjects and f i v e patients with f a m i l i a l LCAT de f i c i e n c y . He found that normal plasma contained 5-8 ug LCAT/ml, a value not s i g n i f i c a n t l y d i f f e r e n t from that reported i n this t h e s i s . The two patients, S.F. and D.H., described i n this thesis were among the f i v e i n d i viduals investigated i n - 60 -Alber's study. The apparent absence of immunochemically detectable LCAT i n the two patients, however, only indicates the absence of an immunoreactive enzyme i n t h e i r plasma. The p o s s i b i l i t y e x i s t s that an immunologically d i s t i n c t and ina c t i v e form of the enzyme may be present i n the patients' plasma, whether f a m i l i a l LCAT deficiency is always due to the complete absence of the enzyme therefore remains unknown (78). There is a general b e l i e f now that heterogeneity of LCAT deficiency e x i s t s (76). Of the five patients that were studied by Albers et a l . , only two (S.F. and D.H.) had a complete lack of immunoreactive LCAT. Two other patients had a LCAT concentration of about 10-15% of that found i n normal plasma. However, these two patients had no measurable enzymatic a c t i v i t y . One patient had a very low protein mass of LCAT ( 1% normal) but enzymatic a c t i v i t y of 10% of normal (39). These re s u l t s suggest that LCAT deficiency may ar i s e from more than one form of mutation. The fact that some patients had no immunoreactive enzyme makes i t less desirable to treat them with p u r i f i e d enzyme as replacement therapy. The r i s k of producing antibody against the enzyme in such patients must be considered. These studies of LCAT d e f i c i e n t patients also show the importance of combining the immunoassay together with f u c t i o n a l assay. Studies of LCAT mass measurement enzymatic a c t i v i t y may be useful i n our future understanding of how LCAT functions i n pathological states such as hypolipidemia, hyper-l i p idemia and l i v e r disease. Results from the study of family members in the LCAT d e f i c i e n t patients indicated that a good c o r r e l a t i o n existed i n the i r plasma quantity of LCAT and t h e i r enzymatic a c t i v i t y (Table X). Subjects GF, DF, RF and LF showed subnormal amount LCAT proteins with a concurrent decrease i n t h e i r e s t e r i f i c a t i o n a c t i v i t y . These individuals have been previously - 61 -reported as heterozygotes and c a r r i e r s of the disease (79). Similar studies were done by Albers using the same samples and comparable r e s u l t s were obtained (personal communication). These findings e s t a b l i s h for the f i r s t time a method for the detection of the heterozygotes. It i s of interest that the heterozygotes among the members of t h i s family also have d i s t i n c t abnormalities i n t h e i r erythrocyte membranes (79,82). These findings may be of importance i n view of the reported r e l a t i o n s h i p between LCAT a c t i v i t y and l i p i d composition of c e l l membrane (83). Unfortunately, the use of e l e c t r o -immunoassay at this stage is not s e n s i t i v e enough to detect meaningful differences among normal subjects. As a r e s u l t , a conclusive c o r r e l a t i o n between the enzyme mass and i t s a c t i v i t y i n normal subjects could not be established. However, the combination of measuring both the' quantity and the a c t i v i t y of the enzyme does look promising as a tool i n the i n v e s t i g a t i o n of LCAT function i n l i p o p r o t e i n metabolism. It is obvious that the electroimmunoassay described i n this thesis has a few drawbacks. The t i t r e of the antiserum was undesirably low, thus l i m i t i n g the number of studies that could be done, and r e s u l t i n g i n a less s e n s i t i v e assay. Yet prolonged immunizations had proven to be undesirable due to the appearance of unwanted contaminants. Another l i m i t a t i o n of this assay i s i t s dependence on antiserum a v a i l a b i l i t y and thus a large scale study at one time may not be f e a s i b l e . One of the solutions to these problems is to improve on the LCAT molecule's immunogenicity. However, since anti-LCAT antibody i s currently available, i t would be more reasonable and time saving to exert future e f f o r t i n the development of some other type of immunoassay which would u t i l i z e less antiserum. Enzyme-linked immunosorbent assay (ELISA) appears to be a good candidate since only small amount of antiserum i s needed and a large - 62 -number of samples can be done at one time. This assay also o f f e r s the advantage that no radioactive isotope is required. In conclusion, the developed electroimmunoassay to human LCAT appears to be a successful method for assay of LCAT protein mass i n plasma. This method is fast and simple and re s u l t s are reproducible. When used i n conjunction with the functional assay, this immunoassay provided strong evidence of autosomal recessive inheritance i n two patients with f a m i l i a l LCAT de f i c i e n c y . Results from the study also substantiated the importance and usefulness i n e s t a b l i s h i n g a r e l a t i o n s h i p between LCAT protein mass and i t s enzymatic a c t i v i t y . Furthermore, success i n the production of antibody against LCAT opens up opportunities for future projects such as the development of an a f f i n i t y chromatography column (to be included i n the p u r i f i c a t i o n procedure), the production of monoclonal antibody (study for isomorphs of LCAT) or immunohistology of LCAT i n organs or tissue s . - 63 -References 1. Sperry, W.M. Cholesterol esterase i n blood. J . B i o l . Chem. I l l , 467, 1935. 2. Glomset, J.A. The mechanism of the plasma cho l e s t e r o l e s t e r i f i c a t i o n reaction plasma f a t t y acid transferase. Biochim. Biophys. Acta 65, 128, 1962. 3. F i e l d i n g , C.J., Shore, V.G. & F i e l d i n g , P.E. A protein cofactor of l e c i t h i n : c h o l e s t e r o l acyltransferase. Biochem. Biophys. Res. 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